Ciera Martinez
September 8, 2014
Pattern 1: Tip to base wave of differentiation (basipetal).
Cell differentation occurring first at the tip.
Pattern 2: Marginal blastozone or marginal meristem, is defined histologically as having dense cells and maintenance of high rates of cell division.
What are the genes expression trends observed in early complex leaf development that would explain differentiation patterning in the leaf?
Isolate tissue
1. longitudinal axis (tip, mid, base)
2. margins compared with all other tissue (rachis and midvein) regions, to perform gene expression analysis.
Conclusion
Genes that are up-regulated in each tissue type compared to the other in same region reflect cell differentiation patterning.
Question: In which regions and at what developmental stage is photosynthetic activity first evident in early developing leaves?
Approach: Chlorophyll a/b binding protein (CAB)::GUS
localization reflects photosynthetic activity.
Approach: Chlorophyll a/b binding protein (CAB)::GUS
localization reflects photosynthetic activity.
Ubiquitous in mature leaves.
Conclusions 1: Early in leaf development (P4 & P5), the rachis and midviein show CAB activity, suggesting these regions are first to start specialized processes such as photosynthesis.
Conclustion 2: The LCM approach for determining gene expression patterns is capable of predicting verifiable expression patterns!
Question 1: Does clustering give similar GO enrichment results to DE analysis?
Question 2: Are there patterns of gene expression that explain margin/rachis identity?
Question 3: Can we get to single interesting genes that may explain differentiation patterning by subsetting to a finer scale?
There are two choices to subset the data:
PC1 (29.2%)
PC2 (20.8%)
PC3 (20.3%)
PC4 (15.6%)
Groups genes with similar expression patterns to clusters or nodes.
Results have been found to be similar to both k-means clustering (Chitwood et al., 2013),but allow greater handling of data in levels, for instance between species or genotypes.
GO Categories
Question 1: Does clustering give similar GO enrichment results to DE analysis?
Yes. Recapitulates DE expression patterns in tissue specific regions.
Question 2: What are the specifc genes that contribute to marginal idenity?
Cluster 17: Up-regulated in rachis compared to margin tissue within each region
Auxin Response 6 - auxin response via expression of auxin regulated genes
gibberellin 2-oxidase - responsive to cytokinin and KNOX activities
ARGONAUTE7 - required for mediolateral expansion in maize (Douglas et al, 2010)
REDUCED STEM BRANCHING 6 - MADS-box transcription factor, mutant flower margins expanded (TAIR mutant lines)
AP2/B3 domain transcription factor - may function as a negative growth regulator
R2R3-MYB TF factor gene - MYB gene involved in cell fate idenity & Lateral Meristem Initiation (Muller et al., 2005)
EMBRYO DEFECTIVE - mutant with enlarged SAM (Cushing et al. 2005)
Solyc00g277510 - Encodes chlorophyll binding protein D1, a part of the photosystem II reaction center core
Solyc02g071000 - Subunit of light-harvesting complex II (LHCII),which absorbs light and transfers energy to the photosynthetic reaction center.
Solyc05g013570 - phototropic-responsive NPH3 family protein
Solyc05g041230 - chloroplast gene encoding a CP43 subunit of the photosystem II reaction center.
Solyc08g066500 - Member of the class III HD-ZIP protein family. Critical for vascular development.
Solyc08g067330 - Encodes lhcb1.1 a component of the LHCIIb light harvesting complex associated with photosystem II.
Mutant is unable to maintain proper auxin foci due to low PIN1 expression levels
GO sequence-specific DNA binding transcription factor activity & transcription factor complex
tf2 specific includes developmental genes YABBY, KNAT6, ARF8, SCARECROW
WT specific includes NAC transcription factor
Still too many genes!
Future : Need to look at larger SOMs for more refined co-expression patterns.
Super SOM: clusters have dimensionality and a separate identity associated with genotype data set, but ultimately, data must be assigned to the same cluster.
Solyc12g006340.1.1 (ARF8):Encodes a member of the auxin response factor family.
Solyc10g076790.1.1 (AUX1): Encodes an auxin influx transporter.
Solyc03g118740.2.1 (PIN1): Auxin efflux.
Solyc09g065820.2.1: DNA binding / transcription factor; cell differentiation.
Solyc09g010780.2.1: Involved in leaf development. Knockout mutants have abnormally shaped leaves.
Solyc02g080260.2.1: Encodes a homeodomain protein that is expressed in the LI layer of the vegetative
Solyc07g018290.2.1 (PLETHERA 1): expressed in young tissues and may specify meristematic or division-competent states. (Wilson et al., 2005).
Solyc06g075850.1.1: Histone H4 : cytochrome P450 monooxygenase. Expressed in cotyledons and leaves
Solyc04g009950.2.1: R2R3- type MYB- encoding genes